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1.
Mol Oncol ; 15(10): 2507-2543, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34515408

RESUMO

Key stakeholders from the cancer research continuum met in May 2021 at the European Cancer Research Summit in Porto to discuss priorities and specific action points required for the successful implementation of the European Cancer Mission and Europe's Beating Cancer Plan (EBCP). Speakers presented a unified view about the need to establish high-quality, networked infrastructures to decrease cancer incidence, increase the cure rate, improve patient's survival and quality of life, and deal with research and care inequalities across the European Union (EU). These infrastructures, featuring Comprehensive Cancer Centres (CCCs) as key components, will integrate care, prevention and research across the entire cancer continuum to support the development of personalized/precision cancer medicine in Europe. The three pillars of the recommended European infrastructures - namely translational research, clinical/prevention trials and outcomes research - were pondered at length. Speakers addressing the future needs of translational research focused on the prospects of multiomics assisted preclinical research, progress in Molecular and Digital Pathology, immunotherapy, liquid biopsy and science data. The clinical/prevention trial session presented the requirements for next-generation, multicentric trials entailing unified strategies for patient stratification, imaging, and biospecimen acquisition and storage. The third session highlighted the need for establishing outcomes research infrastructures to cover primary prevention, early detection, clinical effectiveness of innovations, health-related quality-of-life assessment, survivorship research and health economics. An important outcome of the Summit was the presentation of the Porto Declaration, which called for a collective and committed action throughout Europe to develop the cancer research infrastructures indispensable for fostering innovation and decreasing inequalities within and between member states. Moreover, the Summit guidelines will assist decision making in the context of a unique EU-wide cancer initiative that, if expertly implemented, will decrease the cancer death toll and improve the quality of life of those confronted with cancer, and this is carried out at an affordable cost.

2.
Acta Vet Hung ; 2021 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-34487512

RESUMO

Gestation length (GL) data of dromedary camels were analysed for the period from 2007 to 2018. The database of the largest dairy camel herds (Dubai, United Arab Emirates) was used in this study. The data of 4,084 camels included in the assessment were classified into six ecotypes (Emirati, Emirati cross, Black, Pakistani, Saudi-Sudanese and Saudi cross). The aim of the study was to describe the heritability of GL of camels and the breeding value (BV) of sires for this trait. The genetic parameters of GL were estimated by the General Linear Model method and two Best Linear Unbiased Prediction (BLUP) animal models as well. The mean (±SE) of GL of camels was 384.3 ± 0.2 days. The direct heritability of GL (0.26 ± 0.06-0.36 ± 0.08) was higher than the maternal heritability (0.00 ± 0.05-0.13 ± 0.06) obtained. The maternal permanent environmental effect (0.15 ± 0.05) was similar to the results estimated previously in dromedary camel, but higher than the data reported by relevant sources in other species. Based on the results of this study it can be concluded that the GL of dromedary camels is a species-specific value similar to that in cattle, which is less affected by the maternal influence. Considerable differences (16 days) exist among male dromedaries in their BV for the GL trait.

3.
Free Radic Biol Med ; 176: 222-227, 2021 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-34582996

RESUMO

Protein phosphatase Z1 (Ppz1) has been shown to take part in important physiological functions in fungi including a contribution to virulence of Candida albicans. Although its involvement in the oxidative stress response has also been documented, the exact mechanism of action of its protective effect against oxidative damage remains unknown. By developing a pipeline to analyze the biophysical properties of the cell membrane in fungi, we demonstrate that the plasma membrane of Ppz1-KO Candida albicans displays increased sensitivity to tert-butyl-hydroperoxide-induced oxidative damage. In particular, the response to the oxidizing agent, characterized by increased lipid peroxidation, reduced lipid order, and inhibited lateral mobility of plasma membrane components, is significantly more pronounced in the Ppz1-KO C. albicans strain than in the wild-type counterpart. Remarkably, membrane constituents became almost completely immobile in the phosphatase deletion mutant exposed to oxidative stress. Furthermore, moderately elevated membrane lipid peroxidation accompanied by the aforementioned changes in the biophysical characteristics of the plasma membrane are already detectable in untreated Ppz1-KO cells indicating latent membrane damage even in the absence of oxidative stress. In conclusion, the hypersensitivity of cells lacking Ppz1 to oxidative damage establishes that potential Ppz1 inhibitors may synergize with oxidizing agents in prospective anti-fungal combination therapies.

4.
Virology ; 563: 1-19, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34399236

RESUMO

To further our understanding of the pro-viral roles of the host cytosolic heat shock protein 70 (Hsp70) family, we chose the conserved Arabidopsis thaliana Hsp70-2 and the unique Erd2 (early response to dehydration 2), which contain Hsp70 domains. Based on in vitro studies with purified components, we show that AtHsp70-2 and AtErd2 perform pro-viral functions equivalent to that of the yeast Ssa1 Hsp70. These functions include activation of the tombusvirus RdRp, and stimulation of replicase assembly. Yeast-based complementation studies demonstrate that AtHsp70-2 or AtErd2 are present in the purified tombusvirus replicase. RNA silencing and over-expression studies in Nicotiana benthamiana suggest that both Hsp70-2 and Erd2 are co-opted by tomato bushy stunt virus (TBSV). Moreover, we used allosteric inhibitors of Hsp70s to inhibit replication of TBSV and related plant viruses in plants. Altogether, interfering with the functions of the co-opted Hsp70s could be an effective antiviral approach against tombusviruses in plants.

5.
J Virol ; 95(21): e0107621, 2021 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-34406861

RESUMO

Positive-strand RNA viruses induce the biogenesis of unique membranous organelles called viral replication organelles (VROs), which perform virus replication in infected cells. Tombusviruses have been shown to rewire cellular trafficking and metabolic pathways, remodel host membranes, and recruit multiple host factors to support viral replication. In this work, we demonstrate that tomato bushy stunt virus (TBSV) and the closely related carnation Italian ringspot virus (CIRV) usurp Rab7 small GTPase to facilitate building VROs in the surrogate host yeast and in plants. Depletion of Rab7 small GTPase, which is needed for late endosome and retromer biogenesis, strongly inhibits TBSV and CIRV replication in yeast and in planta. The viral p33 replication protein interacts with Rab7 small GTPase, which results in the relocalization of Rab7 into the large VROs. Similar to the depletion of Rab7, the deletion of either MON1 or CCZ1 heterodimeric GEFs (guanine nucleotide exchange factors) of Rab7 inhibited TBSV RNA replication in yeast. This suggests that the activated Rab7 has proviral functions. We show that the proviral function of Rab7 is to facilitate the recruitment of the retromer complex and the endosomal sorting nexin-BAR proteins into VROs. We demonstrate that TBSV p33-driven retargeting of Rab7 into VROs results in the delivery of several retromer cargos with proviral functions. These proteins include lipid enzymes, such as Vps34 PI3K (phosphatidylinositol 3-kinase), PI4Kα-like Stt4 phosphatidylinositol 4-kinase, and Psd2 phosphatidylserine decarboxylase. In summary, based on these and previous findings, we propose that subversion of Rab7 into VROs allows tombusviruses to reroute endocytic and recycling trafficking to support virus replication. IMPORTANCE The replication of positive-strand RNA viruses depends on the biogenesis of viral replication organelles (VROs). However, the formation of membranous VROs is not well understood yet. Using tombusviruses and the model host yeast, we discovered that the endosomal Rab7 small GTPase is critical for the formation of VROs. Interaction between Rab7 and the TBSV p33 replication protein leads to the recruitment of Rab7 into VROs. TBSV-driven usurping of Rab7 has proviral functions through facilitating the delivery of the co-opted retromer complex, sorting nexin-BAR proteins, and lipid enzymes into VROs to create an optimal milieu for virus replication. These results open up the possibility that controlling cellular Rab7 activities in infected cells could be a target for new antiviral strategies.

6.
Inflamm Bowel Dis ; 2021 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-34427633

RESUMO

BACKGROUND: Patients with Crohn's disease (CD) experience intestinal inflammation. Ontamalimab (SHP647), a fully human immunoglobulin G2 monoclonal antibody against mucosal addressin cell adhesion molecule-1, is a potential novel CD treatment. OPERA II, a multicenter, open-label, phase 2 extension study, assessed the long-term safety and efficacy of ontamalimab in patients with moderate-to-severe CD. METHODS: Patients had completed 12 weeks of blinded treatment (placebo or ontamalimab at 22.5, 75, or 225 mg subcutaneously) in OPERA (NCT01276509) or had a clinical response to ontamalimab 225 mg in TOSCA (NCT01387594). Participants received ontamalimab at 75 mg every 4 weeks (weeks 0-72), then were followed up every 4 weeks for 24 weeks. One-time dose reduction to 22.5 mg or escalation to 225 mg was permitted at the investigator's discretion. The primary end points were safety and tolerability outcomes. Secondary end points included changes in serum drug and biomarker concentrations. Efficacy end points were exploratory, and used non-responder imputation methods. RESULTS: Overall, 149/268 patients completed the study. The most common adverse event leading to study discontinuation was CD flare (19.8%). Two patients died; neither death was considered to be drug related. No dose reductions occurred; 157 patients had their dose escalated. Inflammatory biomarker concentrations decreased. Serum ontamalimab levels were consistent with known pharmacokinetics. Remission rates (Harvey-Bradshaw Index [HBI] ≤ 5; baseline, 48.1%; week 72, 37.3%) and response rates (baseline [decrease in Crohn's Disease Activity Index ≥ 70 points], 63.1%; week 72 [decrease in HBI ≥ 3], 42.5%) decreased gradually. CONCLUSIONS: Ontamalimab was well tolerated; treatment responses appeared to be sustained over 72 weeks.ClinicalTrials.gov ID: NCT01298492.

7.
Int J Mol Sci ; 22(14)2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34299206

RESUMO

Despite the intensive investigation of the molecular mechanism of skeletal muscle hypertrophy, the underlying signaling processes are not completely understood. Therefore, we used an overload model, in which the main synergist muscles (gastrocnemius, soleus) of the plantaris muscle were surgically removed, to cause a significant overload in the remaining plantaris muscle of 8-month-old Wistar male rats. SIRT1-associated pro-anabolic, pro-catabolic molecular signaling pathways, NAD and H2S levels of this overload-induced hypertrophy were studied. Fourteen days of overload resulted in a significant 43% (p < 0.01) increase in the mass of plantaris muscle compared to sham operated animals. Cystathionine-ß-synthase (CBS) activities and bioavailable H2S levels were not modified by overload. On the other hand, overload-induced hypertrophy of skeletal muscle was associated with increased SIRT1 (p < 0.01), Akt (p < 0.01), mTOR, S6 (p < 0.01) and suppressed sestrin 2 levels (p < 0.01), which are mostly responsible for anabolic signaling. Decreased FOXO1 and SIRT3 signaling (p < 0.01) suggest downregulation of protein breakdown and mitophagy. Decreased levels of NAD+, sestrin2, OGG1 (p < 0.01) indicate that the redox milieu of skeletal muscle after 14 days of overloading is reduced. The present investigation revealed novel cellular interactions that regulate anabolic and catabolic processes in the hypertrophy of skeletal muscle.


Assuntos
Cistationina beta-Sintase/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/patologia , Animais , Hipertrofia/genética , Hipertrofia/metabolismo , Hipertrofia/patologia , Masculino , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Proteínas Quinases S6 Ribossômicas/genética , Proteínas Quinases S6 Ribossômicas/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Sirtuínas/antagonistas & inibidores , Sirtuínas/genética , Sirtuínas/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
8.
Theriogenology ; 172: 289-299, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34304044

RESUMO

In this paper, we described the incidence of early pregnancy loss (EPL) both after natural mating and embryo transfer, evaluated risk factors, and summarized the outcome of twin pregnancies throughout gestation in dromedaries under reproductive care. Data were collected over seven breeding seasons at the world's largest camel dairy farm (study 1). In addition, we determined the timing of EPL and monitored serum progesterone (P4) concentration between Days 13-70 of gestation during one breeding season (study 2). In the first study, out of 2970 pregnancies, 507 cases (17.1%) of EPL were diagnosed with transrectal ultrasonography. The rate of EPL after natural mating and embryo transfer was 16.1% (n = 422 out of 2616) and 24.0% (n = 85 out of 354), respectively. Twin pregnancies were detected in 215 cases (7.2% of all gestations), and 57 of those (26.5%) underwent complete EPL. Almost half of the early losses (n = 243; 47.9%) occurred before 30 d of gestation. Another 43.2% (n = 219) of EPL was diagnosed during the next month, and 8.9% (n = 45) occurred after 60 d of gestation. Multivariable mixed effects logistic regression models revealed that the breeding season (year) and twin pregnancy were the most important exposure variables affecting the rate of EPL (P < 0.001). The effect of some male camels was also demonstrated while other factors, such as type of breeding, age category, month of mating, breed/ecotype and reproductive history did not prove to have a significant influence. In the second study, the overall rate of EPL was 24.5% (n = 34 of 139). There was no difference in the incidence of EPL between ET recipient (24.2%, n = 23 of 95) and mated (25%, n = 11 of 44) camels. Weekly rate of EPL ranged from 0.9% to 4.8% with a decreasing tendency, and approx. 41% of the animals (n = 14 of 34) had some ultrasonographic signs of impending EPL 1 week before the final diagnosis. Mean serum P4 concentration in camels with subsequent EPL was 5.3 ± 0.1 ng/ml compared to 5.6 ± 0.04 ng/ml in normal pregnant dromedaries. Day of gestation and future EPL influenced serum P4 levels (P < 0.001) with an interaction between the two fixed factors (P < 0.05). At the time of the final diagnosis of EPL, mean serum P4 concentration was 2.8 ± 0.44 ng/ml. Although twinning had an unfavorable prognosis with a total pregnancy loss of 36.7%, it was not entirely detrimental for the final outcome of gestation as two-thirds of twin pregnancies (n = 136 out of 212) resulted in the birth of a live calf.


Assuntos
Aborto Animal , Camelus , Aborto Animal/epidemiologia , Animais , Transferência Embrionária/veterinária , Feminino , Incidência , Masculino , Parto , Gravidez
9.
J Acoust Soc Am ; 149(6): 4298, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34241461

RESUMO

Rayleigh waves are well known to attenuate due to scattering when they propagate over a rough surface. Theoretical investigations have derived analytical expressions linking the attenuation coefficient to statistical surface roughness parameters, namely, the surface's root mean squared height and correlation length and the Rayleigh wave's wavenumber. In the literature, three scattering regimes have been identified-the geometric (short wavelength), stochastic (short to medium wavelength), and Rayleigh (long wavelength) regimes. This study uses a high-fidelity two-dimensional finite element (FE) modelling scheme to validate existing predictions and provide a unified approach to studying the problem of Rayleigh wave scattering from rough surfaces as the same model can be used to obtain attenuation values regardless of the scattering regime. In the Rayleigh and stochastic regimes, very good agreement is found between the theory and FE results both in terms of the absolute attenuation values and for asymptotic power relationships. In the geometric regime, power relationships are obtained through a combination of dimensional analysis and FE simulations. The results here also provide useful insight into verifying the three-dimensional theory because the method used for its derivation is analogous.

10.
Phytochemistry ; 190: 112851, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34217043

RESUMO

Fungal endophytes are remarkable sources of biologically active metabolites of ecological and pharmacological significance. In this study, fungal isolates producing yellow pigments and originating from grass roots, were identified as the recently described grass root colonizing dark septate endophyte (DSE), Flavomyces fulophazii (Periconiaceae, Pleosporales). While analyzing the metabolite composition of 17 isolates of this fungus, 11 previously undescribed compounds, including four tetramic acids (dihydroxyvermelhotin, hydroxyvermelhotin, methoxyvermelhotin, oxovermelhotin), and seven chlorinated azaphilones (flavochlorines A-G), together with the known tetramic acid vermelhotin, were tentatively identified by high performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS). Among them, flavochlorine A, flavochlorine G, hydroxyvermelhotin and vermelhotin could be isolated by preparative HPLC, thus their structures were also confirmed by nuclear magnetic resonance (NMR) spectroscopy. Vermelhotin was found to be the main compound, reaching its maximum level of 5.5 mg/g in the in vitro cultures of a selected F. fulophazii isolate. A significant amount of vermelhotin was isolated by preparative HPLC from these cultures (4.8 mg from 1.0 g lyophilized culture), confirming the practical utility of F. fulophazii in high-yield vermelhotin production. The main compounds of this endophyte expressed no activity in standardized plant bioassays (i.e., in the Lactuca sativa seed germination and Lemna minor growth tests). An antiproliferative study of the isolated compounds confirmed moderate activity of vermelhotin against a panel of twelve cancer cell lines, with IC50 ranges of 10.1-37.0 µM, without inhibiting the non-cancer Vero cells, suggesting its selectivity towards cancers.


Assuntos
Ascomicetos , Espectrometria de Massas em Tandem , Animais , Benzopiranos , Chlorocebus aethiops , Endófitos , Pigmentos Biológicos , Pirrolidinonas , Células Vero
11.
PLoS Pathog ; 17(6): e1009680, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34161398

RESUMO

Positive-strand (+)RNA viruses take advantage of the host cells by subverting a long list of host protein factors and transport vesicles and cellular organelles to build membranous viral replication organelles (VROs) that support robust RNA replication. How RNA viruses accomplish major recruitment tasks of a large number of cellular proteins are intensively studied. In case of tomato bushy stunt virus (TBSV), a single viral replication protein, named p33, carries out most of the recruitment duties. Yet, it is currently unknown how the viral p33 replication protein, which is membrane associated, is capable of the rapid and efficient recruitment of numerous cytosolic host proteins to facilitate the formation of large VROs. In this paper, we show that, TBSV p33 molecules do not recruit each cytosolic host factor one-by-one into VROs, but p33 targets a cytosolic protein interaction hub, namely Rpn11, which interacts with numerous other cytosolic proteins. The highly conserved Rpn11, called POH1 in humans, is the metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates. However, TBSV takes advantage of a noncanonical function of Rpn11 by exploiting Rpn11's interaction with highly abundant cytosolic proteins and the actin network. We provide supporting evidence that the co-opted Rpn11 in coordination with the subverted actin network is used for delivering cytosolic proteins, such as glycolytic and fermentation enzymes, which are readily subverted into VROs to produce ATP locally in support of VRO formation, viral replicase complex assembly and viral RNA replication. Using several approaches, including knockdown of Rpn11 level, sequestering Rpn11 from the cytosol into the nucleus in plants or temperature-sensitive mutation in Rpn11 in yeast, we show the inhibition of recruitment of glycolytic and fermentation enzymes into VROs. The Rpn11-assisted recruitment of the cytosolic enzymes by p33, however, also requires the combined and coordinated role of the subverted actin network. Accordingly, stabilization of the actin filaments by expression of the Legionella VipA effector in yeast and plant, or via a mutation of ACT1 in yeast resulted in more efficient and rapid recruitment of Rpn11 and the selected glycolytic and fermentation enzymes into VROs. On the contrary, destruction of the actin filaments via expression of the Legionella RavK effector led to poor recruitment of Rpn11 and glycolytic and fermentation enzymes. Finally, we confirmed the key roles of Rpn11 and the actin filaments in situ ATP production within TBSV VROs via using a FRET-based ATP-biosensor. The novel emerging theme is that TBSV targets Rpn11 cytosolic protein interaction hub driven by the p33 replication protein and aided by the subverted actin filaments to deliver several co-opted cytosolic pro-viral factors for robust replication within VROs.


Assuntos
Citoesqueleto de Actina/metabolismo , Endopeptidases/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Tombusvirus/fisiologia , Replicação Viral/fisiologia , Citosol/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo
12.
Nat Commun ; 12(1): 3927, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34168142

RESUMO

Quantum-mechanical methods are used for understanding molecular interactions throughout the natural sciences. Quantum diffusion Monte Carlo (DMC) and coupled cluster with single, double, and perturbative triple excitations [CCSD(T)] are state-of-the-art trusted wavefunction methods that have been shown to yield accurate interaction energies for small organic molecules. These methods provide valuable reference information for widely-used semi-empirical and machine learning potentials, especially where experimental information is scarce. However, agreement for systems beyond small molecules is a crucial remaining milestone for cementing the benchmark accuracy of these methods. We show that CCSD(T) and DMC interaction energies are not consistent for a set of polarizable supramolecules. Whilst there is agreement for some of the complexes, in a few key systems disagreements of up to 8 kcal mol-1 remain. These findings thus indicate that more caution is required when aiming at reproducible non-covalent interactions between extended molecules.


Assuntos
Modelos Químicos , Benchmarking , Benzeno/química , Bases de Dados de Compostos Químicos , Difusão , Ligação de Hidrogênio , Método de Monte Carlo , Piridinas/química , Teoria Quântica , Eletricidade Estática , Uracila/química , Água/química
13.
Chemistry ; 27(42): 11005-11014, 2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-33999467

RESUMO

Lysine methylation can modify noncovalent interactions by altering lysine's hydrophobicity as well as its electronic structure. Although the ramifications of the former are documented, the effects of the latter remain largely unknown. Understanding the electronic structure is important for determining how biological methylation modulates protein-protein binding, and the impact of artificial methylation experiments in which methylated lysines are used as spectroscopic probes and protein crystallization facilitators. The benchmarked first-principles calculations undertaken here reveal that methyl-induced polarization weakens the electrostatic attraction of amines with protein functional groups - salt bridges, hydrogen bonds and cation-π interactions weaken by as much as 10.3, 7.9 and 3.5 kT, respectively. Multipole analysis shows that weakened electrostatics is due to the altered inductive effects, which overcome increased attraction from methyl-enhanced polarizability and dispersion. Due to their fundamental nature, these effects are expected to be present in many cases. A survey of methylated lysines in protein structures reveals several cases in which methyl-induced polarization is the primary driver of altered noncovalent interactions; in these cases, destabilizations are found to be in the 0.6-4.7 kT range. The clearest case of where methyl-induced polarization plays a dominant role in regulating biological function is that of the PHD1-PHD2 domain, which recognizes lysine-methylated states on histones. These results broaden our understanding of how methylation modulates noncovalent interactions.


Assuntos
Lisina , Proteínas , Ligação de Hidrogênio , Lisina/metabolismo , Ligação Proteica , Proteínas/metabolismo , Eletricidade Estática
14.
Cell Rep ; 35(7): 109137, 2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34010645

RESUMO

Oncogenic histone lysine-to-methionine mutations block the methylation of their corresponding lysine residues on wild-type histones. One attractive model is that these mutations sequester histone methyltransferases, but genome-wide studies show that mutant histones and histone methyltransferases often do not colocalize. Using chromatin immunoprecipitation sequencing (ChIP-seq), here, we show that, in fission yeast, even though H3K9M-containing nucleosomes are broadly distributed across the genome, the histone H3K9 methyltransferase Clr4 is mainly sequestered at pericentric repeats. This selective sequestration of Clr4 depends not only on H3K9M but also on H3K14 ubiquitylation (H3K14ub), a modification deposited by a Clr4-associated E3 ubiquitin ligase complex. In vitro, H3K14ub synergizes with H3K9M to interact with Clr4 and potentiates the inhibitory effects of H3K9M on Clr4 enzymatic activity. Moreover, binding kinetics show that H3K14ub overcomes the Clr4 aversion to H3K9M and reduces its dissociation. The selective sequestration model reconciles previous discrepancies and demonstrates the importance of protein-interaction kinetics in regulating biological processes.

15.
Curr Opin Virol ; 48: 30-41, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33845410

RESUMO

Positive-strand RNA viruses depend on intensive manipulation of subcellular organelles and membranes to create unique viral replication organelles (VROs), which represent the sites of robust virus replication. The host endomembrane-based protein-trafficking and vesicle-trafficking pathways are specifically targeted by many (+)RNA viruses to take advantage of their rich resources. We summarize the critical roles of co-opted endoplasmic reticulum subdomains and associated host proteins and COPII vesicles play in tombusvirus replication. We also present the surprising contribution of the early endosome and the retromer tubular transport carriers to VRO biogenesis. The central player is tomato bushy stunt virus (TBSV), which provides an outstanding system based on the identification of a complex network of interactions with the host cells. We present the emerging theme on how TBSV uses tethering and membrane-shaping proteins and lipid modifying enzymes to build the sophisticated VRO membranes with unique lipid composition.

16.
J Chem Theory Comput ; 17(5): 2886-2905, 2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-33819030

RESUMO

A linear-scaling local second-order Møller-Plesset (MP2) method is presented for high-spin open-shell molecules based on restricted open-shell (RO) reference functions. The open-shell local MP2 (LMP2) approach inherits the iteration- and redundancy-free formulation and the completely integral-direct, OpenMP-parallel, and memory and disk use economic algorithms of our closed-shell LMP2 implementation. By utilizing restricted local molecular orbitals for the demanding integral transformation step and by introducing a novel long-range spin-polarization approximation, the computational cost of RO-LMP2 approaches that of closed-shell LMP2. Extensive benchmarks were performed for reactions of radicals, ionization potentials, as well as spin-state splittings of carbenes and transition-metal complexes. Compared to the conventional MP2 reference for systems of up to 175 atoms, local errors of at most 0.1 kcal/mol were found, which are well below the intrinsic accuracy of MP2. RO-LMP2 computations are presented for challenging protein models of up to 601 atoms and 11 000 basis functions, which involve either spin states of a complexed iron ion or a highly delocalized singly occupied orbital. The corresponding runtimes of 9-15 h obtained with a single, many-core CPU demonstrate that MP2, as well as spin-scaled MP2 and double-hybrid density functional methods, become widely accessible for open-shell systems of unprecedented size and complexity.

17.
Sci Adv ; 7(17)2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33883133

RESUMO

The recent report by Fan et al alleged that the ProPerDP method is inadequate for the detection of protein persulfidation. Upon careful evaluation of their work, we conclude that the claim made by Fan et al is not supported by their data, rather founded in methodological shortcomings. It is understood that the ProPerDP method generates a mixture of cysteine-containing and non-cysteine-containing peptides. Instead, Fan et al suggested that the detection of non-cysteine-containing peptides indicates nonspecific alkylation at noncysteine residues. However, if true, then such peptides would not be released by reduction and therefore not appear as products in the reported workflow. Moreover, the authors' biological assessment of ProPerDP using Escherichia coli mutants was based on assumptions that have not been confirmed by other methods. We conclude that Fan et al did not rigorously assess the method and that ProPerDP remains a reliable approach for analyses of protein per/polysulfidation.

18.
Virology ; 559: 15-29, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33799077

RESUMO

Positive-strand RNA viruses build viral replication organelles (VROs) with the help of co-opted host factors. The energy requirement of intensive viral replication processes is less understood. Previous studies on tomato bushy stunt virus (TBSV) showed that tombusviruses hijack two ATP-producing glycolytic enzymes to produce ATP locally within VROs. In this work, we performed a cDNA library screen with Arabidopsis thaliana proteins and the TBSV p33 replication protein. The p33 - plant interactome contained highly conserved glycolytic proteins. We find that the glycolytic Hxk2 hexokinase, Eno2 phosphopyruvate hydratase and Fba1 fructose 1,6-bisphosphate aldolase are critical for TBSV replication in yeast or in a cell-free replicase reconstitution assay. The recruitment of Fba1 is important for the local production of ATP within VROs. Altogether, our data support the model that TBSV recruits and compartmentalizes possibly most members of the glycolytic pathway. This might allow TBSV to avoid competition with the host for ATP.

19.
Br J Pharmacol ; 178(18): 3667-3681, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33908640

RESUMO

BACKGROUND AND PURPOSE: Cell penetrating peptides are promising tools for delivery of cargo into cells, but factors limiting or facilitating their cellular uptake are largely unknown. We set out to study the effect of the biophysical properties of the cell membrane on the uptake of penetratin, a cell penetrating peptide. EXPERIMENTAL APPROACH: Using labelling with pH-insensitive and pH-sensitive dyes, the kinetics of cellular uptake and endo-lysosomal escape of penetratin were studied by flow cytometry. KEY RESULTS: We report that escape of penetratin from acidic endo-lysosomal compartments is retarded compared with its total cellular uptake. The membrane dipole potential, known to alter transmembrane transport of charged molecules, is shown to be negatively correlated with the concentration of penetratin in the cytoplasmic compartment. Treatment of cells with therapeutically relevant concentrations of atorvastatin, an inhibitor of HMG-CoA reductase and cholesterol synthesis, significantly increased endosomal escape of penetratin in two different cell types. This effect of atorvastatin correlated with its ability to decrease the membrane dipole potential. CONCLUSION AND IMPLICATIONS: These results highlight the importance of the dipole potential in regulating cellular uptake of cell penetrating peptides and suggest a clinically relevant way of boosting this process.

20.
Mol Oncol ; 15(5): 1277-1288, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33734563

RESUMO

There is a persistent variation in cancer outcomes among and within European countries suggesting (among other causes) inequalities in access to or delivery of high-quality cancer care. European policy (EU Cancer Mission and Europe's Beating Cancer Plan) is currently moving towards a mission-oriented approach addressing these inequalities. In this study, we used the quantitative and qualitative data of the Organisation of European Cancer Institutes' Accreditation and Designation Programme, relating to 40 large European cancer centres, to describe their current compliance with quality standards, to identify the hallmarks common to all centres and to show the distinctive features of Comprehensive Cancer Centres. All Comprehensive Cancer Centres and Cancer Centres accredited by the Organisation of European Cancer Institutes show good compliance with quality standards related to care, multidisciplinarity and patient centredness. However, Comprehensive Cancer Centres on average showed significantly better scores on indicators related to the volume, quality and integration of translational research, such as high-impact publications, clinical trial activity (especially in phase I and phase IIa trials) and filing more patents as early indicators of innovation. However, irrespective of their size, centres show significant variability regarding effective governance when functioning as entities within larger hospitals.

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